Shigella flexneri and S. sonnei and representing a major global health risk, particularly in developing countries. Bacterial O-antigen (OAg) is the primary target of the host immune response and modifications of its oligosaccharide units, including O-acetylation, are responsible for the variability among the circulating S. flexneri serotypes. No vaccines are widely available against shigellosis and the understanding of the immunogenicity induced by the OAg is fundamental for the design of a vaccine that could cover the most prevalent Shigella serotypes. To understand whether a different O-acetylation pattern could influence the immune response elicited by S. flexneri OAg, we employed as a vaccine technology GMMA purified from S. flexneri 2a and 1b strains that were easily engineered to obtain differently O-acetylated OAg. Resulting GMMA were tested in mice, demonstrating not only no major impact of O-acetyl decorations on the immune response elicited by the two OAg against the homologous strains, but also that the O-acetylation of the Rhamnose III residue (O-factor 9), shared among serotypes 1b, 2a and 6, does not induce cross-reactive antibodies against these serotypes. This work contributes to the optimization of vaccine design against Shigella, providing indication about the ability of shared epitopes to elicit broad protection against S. flexneri serotypes and supporting the identification of critical quality attributes of OAg-based vaccines.Shigellosis is a diarrheal disease caused prevalently by  Shigella infections are among the top causes of moderate to severe diarrhea throughout the world, causing approximately 112 million cases of which a great percentage is represented by children under 5 years of age .,18.S. flS. flexneri and the antigenicity that resulted from this modification. PCR screening of the oacB gene in 730 clinical isolates of S. flexneri demonstrated that the oacB-mediated 3/4-O-acetylation is widespread in serotypes 1a, 1b, 2a, and Y. Among them, 1a, 1b, and 2a are the predominant serotypes in China and other Asian countries, and the high 3/4-O-acetylation rates (>94%) found in these serotypes suggest that the presence of O-factor 9 may play a role in their preferable dissemination. However further studies are necessary to elucidate if this OAg modification contributes to the virulence of S. flexneri. Wang and colleagues studied the distribution of the 3/4-O-acetylation in S. flexneri serotype 6, despite its genetic determinant was found in the oacC gene (72% homologous to oacB) [O-factor 9 has also been reported in to oacB) . S. flexneri OAg. It has been reported that a S. flexneri 2a OAg conjugate vaccine induced protection against S. flexneri serotype 6 in humans and the O-factor 9 was suggested as the structural basis for the observed cross-reactivity between S. flexneri 2a and S. flexneri 6 LPS [There are no direct evidence in the current literature about the role of O-acetylation in the immune response induced by ri 6 LPS ,22.S. flexneri OAg, in particular for S. flexneri 2a and 1b, two of the most prevalent S. flexneri serotypes, and the impact that OAc epitopes can have in the cross-recognition of homologous and heterologous S. flexneri serotypes. We used the generalized modules for membrane antigens (GMMA) technology to rapidly generate different OAg structures with the desired features. GMMA are outer membrane vesicles naturally released from genetically engineered Gram-negative bacteria, having the great advantage to display the OAg in its natural outer membrane context. Shigella bacteria are mutated in order to increase vesicles formation, through the deletion of the tolR gene [htrB or msbB genes [Shigella [tolR S. flexneri 1b and 2a strains, we generated knock-out mutants of S. flexneri 2a for the O-acetyltransferases genes oacB and oacD and of S. flexneri 1b for the O-acetyltransferase gene oacB. GMMA obtained from these strains were used for immunogenicity studies in mice. Notably, OAc on RhaI of S. flexneri 1b was not altered as its removal would convert the strain to serotype 1a.In this study we aim to verify the criticality of O-acetylation pattern for olR gene . PotentibB genes . For theShigella ,27,28,29Shigella flexneri.The results from this study will contribute to the design of cross-protective OAg-based vaccines against S. flexneri 2a and S. flexneri 1b were acquired from Public Health England (PHE) and engineered to obtain the different mutants. The null mutations were obtained by replacing the genes of interest with an antibiotic resistance cassette by homologous recombination using the lambda red recombineering system [tolR background to generate bacterial strains to be used for serum bactericidal assay (SBA) and GMMA purification, respectively. Absence of O-acetyltransferase genes was confirmed by PCR and the phenotype of each mutant was verified by H1 NMR analysis of OAg extracted from the resulting GMMA, as previously described [g system . The lisescribed ,32. S. flexneri 2a and 1b GMMA were produced and purified as previously described [2PO4, 5% CH3CN, Tosoh Bioscience, Tokyo, Japan) with differential refractive index (dRI) detection, using dextrans as the standards to estimate the molecular size distribution as previously described [1H-NMR analysis measured with a Bruker AvanceIII 400 spectrometer at 400 MHz and 323 K.escribed . GMMA toescribed ,34. The escribed ,33. OAg S. flexneri 2a and 1b, differing for O-acetylation pattern, were tested in mice. Female, five-week-old wild-type CD1 mice were immunized intraperitoneally (IP) with 200 µL of vaccine containing 0.005, 0.05, or 0.5 µg of OAg at day 0 and 28. Single sera were collected at day 27 and 42. All animal studies were performed at GSK ARC Animal Care Facility under the animal project 526/2020-PR 26/05/2020 approved by the Italian Ministry of Health. The studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EEC, the GSK policy on the Care, Welfare and Treatment of Animals, and local animal welfare guidelines under Italian authorization. All sera were analyzed by ELISA for anti-S. flexneri OAg total IgG using wild type S. flexneri 1b and 2a OAg as plate-coating antigens . All coating antigens were purified in house as previously described [S. flexneri 1a, 1b, 2a, 2b, or 6 wild-type or mutant strains, as previously described [GMMA obtained from escribed . Single escribed  against escribed .Analysis was performed using GraphPad Prism 7 . The Kruskal–Wallis analysis with post-hoc Dunn’s test was used to compare multiple groups. Dose–response relationships were evaluated through Spearman’s rank correlation. The parallelism of dose–response curves was assessed by the parallel line method: when the slopes of the curves obtained by log-transforming ELISA or SBA results vs. log transformed antigen doses were not significantly different from each other, comparison of the Y-intercepts was performed.S. flexneri 1b and 2a strains were genetically manipulated to obtain overblebbling phenotypes through knock-out of the tolR gene. With the aim to verify the impact of OAg O-acetylation on the immune response induced in mice, genes coding for O-acetyltransferases were also removed and GMMA producing strains differing for the OAg O-acetylation pattern were obtained, as shown in S. flexneri strain and analytically characterized , around 50% for the medium MW (MMW), and around 35% for the low MW (LMW) OAg (this peak corresponds to core only and core plus few OAg repeats). Moreover, the two S. flexneri 1b strains resulted to have very similar OAg populations: 65% and 35% for the MMW and LMW, respectively.The OAg was extracted from each GMMA and analyzed through HPLC-SEC to determine the OAg molecular weight (MW) distribution. Importantly, introduced mutations did not impact OAg size. Indeed, the four strains of 1H NMR analysis. S. flexneri 2a wild type (wt) OAg was 71% O-acetylated on RhaIII (position 3/4) and 56% O-acetylated on GlcNAc. S. flexneri 2a OAg from strains lacking oacB or oacD were 65% and 66% O-acetylated on GlcNAc and RhaIII, respectively. As expected, no signals related to OAg OAc were detected in the 1H NMR spectrum for the double knock-out mutant ΔoacB/ΔoacD of S. flexneri 2a, confirming complete OAg de-O-acetylation. S. flexneri 1b wt OAg was 63% O-acetylated on RhaI and 80% O-acetylated on RhaIII. The OAg from the strain lacking oacB was 79% O-acetylated on RhaI and not O-acetylated on RhaIII, as expected  and mice were immunized intraperitoneally twice at 4-week intervals.Shigella wt fully O-acetylated OAg as coating antigen for each S. flexneri serotype. For both S. flexneri 2a and 1b, the anti-OAg IgG response induced in mice was not impacted by the OAg O-acetylation pattern, as the anti-OAg IgG obtained by immunizing with the differently O-acetylated GMMA was not statistically different neither at day 27 nor at day 42 (Mice sera obtained after first (day 27) and second immunization (day 42) were analyzed through ELISA using t day 42 . These dShigella strain. L-SBA data confirmed the results obtained by ELISA and proved that the functional activity of sera increased against each GMMA was comparable regardless the presence of OAc groups . In particular, for what concerns S. flexneri 2a GMMA resulted in high bactericidal titers against S. flexneri 1a and 2b (IC50 103–105), while relatively low titers were measured against S. flexneri 1b and 6 (IC50 < 103). However, the presence/absence of OAc on S. flexneri 2a OAg did not make any difference in the functionality of the antibodies raised, indicating that common OAg OAc did not play a role in inducing cross-reactive antibodies across the heterologous S. flexneri strains tested. SBA results, reported in S. flexneri 1b GMMA were tested in SBA against S. flexneri 1a, 2a, and 6 that also present OAc on RhaIII. Serum bactericidal killing of S. flexneri 1a was high (IC50~105), whereas low IC50 titers were measured when sera were tested against S. flexneri 6 and almost no killing was observed in the case of S. flexneri 2a. These results were independent from the presence of O-acetylation on S. flexneri 1b OAg.Similarly, polyclonal sera obtained with Shigella is a major cause of morbidity worldwide and antimicrobial resistance makes the development of a widely accessible vaccine an even higher global health priority [Shigella is related to the high number of serotypes causing the disease. In response to this need, multi-component vaccines are currently under development to ensure broad protection against Shigella [priority . The majShigella ,37.Salmonella Typhi and Salmonella Paratyphi A, where the Vi and OAg immunogenicity were related to their degree of O-acetylation [Streptococcus pneumoniae strains [It is known that structural characteristics of saccharide antigens, including O-acetylation, can play a major role on the immune response elicited and can be critical for the design of highly effective vaccines . This istylation ,40. On t strains , howeverS. flexneri OAg. This can be important not only to assess the protective role against homologous strains, but also against heterologous serotypes sharing common epitopes due to such modifications. Indeed, cross-reactivity could help to reduce the complexity of a Shigella vaccine composition. Few preclinical data have been collected so far on the role that O-acetylation can have on the immune response elicited by S. flexneri 2a and 1b OAg with different O-acetylation patterns by knocking-out the responsible genes, similarly to what has already been reported to obtain different OAg size [GMMA represent a powerful technology to facilitate this kind of investigation, besides being a promising vaccine approach . Indeed,OAg size .Shigella pathogenesis and immunogenicity has never been clarified so far. In this study we looked at the role of S. flexneri 2a and 1b OAg O-acetylation in driving an immune response in mice, when delivered with GMMA. In particular, the aim was to verify the criticality of O-acetylation occurring in two positions of S. flexneri 2a OAg, on RhaIII and on GlcNAc, and on RhaIII of S. flexneri 1b in generating a broad and functional anti-OAg immune response. The impact that OAg O-acetylation has on several aspects of the S. flexneri 2a OAg and the OAc on RhaIII of S. flexneri 1b OAg are not critical for the generation of functional antibodies against homologous or heterologous strains.The data obtained suggested that the OAc on RhaIII or/and GlcNAc of S. flexneri 6 OAg O-acetylation has little effect on OAg conformation and hence may not be essential for the antigenicity of serotype 6. This was corroborated by a similar in vivo study in mice, using S. flexneri 6 GMMA as O-antigen delivery systems, that shows that O-acetylation does not have an impact on the immune response elicited by the serotype 6 O-antigen [S. flexneri 2a OAg to five protective mIgGs showed that none of the acetates affect significantly the antibody recognition [S. flexneri 2a conjugate vaccine was able to elicit in humans cross-reactive antibodies against S. flexneri 6 and is reasonable to attribute this result to the O-factor 9 (OAc of RhaIII), the only epitope that these two OAg structures have in common, although no cross-reactivity was observed from studies done in the mouse model, as also confirmed by our data, independently from the O-acetylation pattern [Shigella serotypes, besides O-acetylation, also OAg glucosylation may play a role in generating conformational epitopes shared among different OAg structures and this aspect deserves further investigation.These results are in line with a recent publication in which we showed that -antigen . Moreoveognition . However pattern . Moreove pattern . This chShigella OAg-based vaccines are being tested in clinical trials and analysis of the sera for their bactericidal activity against a panel of different serotypes will allow to confirm or disprove preclinical data and help also clarifying the role of O-acetylation in the immune response elicited by Shigella OAg.Not necessarily data in mice will be predictive of data in humans, but the systematic study performed here allowed to collect a certain data package that will be corroborated from data from clinical trials soon. Different


The 2019 coronavirus disease (COVID-19) has become a global health crisis. CT chest is considered as an important investigation for early diagnosis as well as assessment of severity of COVID-19 pneumonia. Several articles reported that there is a correlation between ABO blood group system and susceptibility as well as prognosis of the disease. In our study we correlated the CT severity score (CT-SS) and the ABO blood group in patients with COVID-19 infection. This study involved 547 symptomatic patients with pathologically proven COVID-19 infection (positive PCR); non contrast CT chest was done for all cases and CT severity score (CT-SS) was calculated followed by its correlation with the patients’ ABO blood group. Aim of the work was to evaluate the relation between CT-SS and the ABO blood groups in Egyptian patients with COVID-19 infection.n = 153; 28%) was 13.7 (moderate severity), while in patients with blood group O  the mean CT-SS was 6.7 (mild severity). In blood group B patients  the mean CT-SS was 9.1 (mild to moderate severity) and in blood group AB patients  the mean CT-SS was 9.7 (mild to moderate severity).The mean CT-SS in patients with blood group A patients (COVID-19 patients with blood group A are more prone to aggressive CT findings (higher CT-SS) and consequently may be susceptible to increased risk of mortality compared to the patients with other blood groups; however, patients with blood group O are suggested to have the least CT-SS and appear to be relatively protected. COVID-19 pneumonia is a rapidly spreading acute respiratory syndrome –4.At present, the diagnosis of COVID-19 depends on real time reverse transcription polymerase chain reaction (RT-PCR); yet it has some limitations that may cause false negative results. High resolution CT examination is a non-invasive, simple and rapid procedure that can be used as a screening tool for suspected patients as well as provide an objective assessment about the extension of the lung disease and consequently assess the disease burden. CT manifestations are similar to those seen in viral pneumonias, with multifocal ground-glass opacities and consolidation in a peripheral sub-pleural distribution being the most common findings. CT severity score (CT-SS) of the chest is considered as a semiquantitative indicator of lung affection in COVID-19 to assess the disease burden –8.The human ABO blood group system includes four blood types, namely, A, B, AB, and O. Many studies have found that the ABO blood group system plays an important role in various infectious and non-infectious human diseases. Differences in the blood group antigens located on the surface of human red blood cells can increase or decrease host susceptibility to many infections as it can facilitate intracellular uptake, signal transmission, or adhesion and modify the immune response to infection. Because COVID-19 is a new virus, it is unclear whether the ABO blood groups affect severity of COVID-19 disease or not .This study aimed to assess the relation between CT-SS and the ABO blood groups in Egyptian patients with COVID-19 infection.All our patients were referred from the chest clinics with positive RT-PCR for COVID-19 infection. CT chest without contrast using a Siemens 16 slice , was done for our 547 symptomatic patients , to detect if there was pulmonary involvement by COVID-19 infection and the CT severity scoring was calculated, throughout a period extending from May 2020 to December 2020. A radiologist with 20 years of experience in CT chest imaging performed the CT image analysis. All individuals were subjected to clinical and laboratory assessments. Clinical features involved age, gender and disease symptoms such as fever, dry cough, dyspnea, anosmia, ageusia, fatigue and diarrhea. Laboratory findings included ABO blood group test, CBC, CRP, ESR, LDH, D dimer, S. ferritin. An informed consent was obtained from all participants in this study. Confidentiality of all patients’ data was guaranteed. All information was obtained and analyzed with the Excel program.Symptomatic patients with positive PCR examination.CT examination was done from the 5th to 13th days after onset of symptoms to detect the most diagnostic CT findings during the progressive and peak stages.Patients with lung masses, history of interstitial lung disease, previous chest surgery, TB or pleural disease.Patients with co-morbidities including those with hepatic and renal impairment, heart failure, uncontrolled diabetes and patients with malignancy.Each lobe could be awarded a CT score from 0 to 5, depending on the percentage of t involved lobe: score 0—0% involvement; score 1—less than 5% involvement; score 2—5 to 25% involvement; score 3—26 to 49% involvement; score 4—50 to 75% involvement; score 5—greater than 75% involvement. The overall CT score was the sum of the points from each lobe and ranges from 0 to 25 points with sensitivity and specificity of 80.0% and 82.8%, respectively .p values of < 0.1 were used to denote statistical significance.The Statistical Package for the Social Sciences  was utilized in the data manipulation and significance testing. The patients were classified into three groups according to their CT-SS: those with CT-SS of less than 9 were considered mild, whereas those with CT-SS of 9–15 were considered moderate and those with CT-SS of more than 15 were considered severe. The Chi-square correlation analysis was conducted, and n = 153; 28%) was 13.7 (moderate severity), while in blood group AB patients  the mean CT-SS was 9.7 (mild to moderate severity) and in blood group B patients  the mean CT-SS was 9.1 (mild to moderate severity). In patients with blood group O  the mean CT-SS was 6.7 (mild severity). The mean CT-SS for all patients was 9.9.During the study period, 547 symptomatic patients with positive PCR test for COVID-19  underwent lab investigations including blood group test, CBC, CRP, ESR, LDH, D dimer, S. ferritin. High resolution CT chest without contrast was done for all of them and the CT-SS was calculated. Of these, the mean CT-SS in patients with blood group A patients  which was matching with the average percentage among the Egyptian population.


Kassina senegalensis. Using site-specific sequence enrichment with a motif “glycine-lysine” that frequently occurs in ranid frog temporin peptides, a series of QUB-1641 analogues were synthesized, and effects on selected bioactivities were studied. The greatest activity enhancement was obtained when the “glycine-lysine” motif was located at the eighth and ninth position as in QUB-1570.QUB-1570 had a broader antibacterial spectrum than QUB-1641, and was eight-fold more potent. Moreover, QUB-1570 inhibited S. aureus biofilm most effectively, and significantly enhanced the viability of insect larvae infected with S. aureus. When the “glycine-lysine” motif of QUB-1570 was substituted to reduce the helix ratio and positive charge, the antibacterial activities of these synthetic analogues decreased. These data revealed that the “glycine-lysine” motif at positions 8 and 9 had the greatest enhancing effect on the antibacterial properties of QUB-1570 through increasing positive charge and helix content. This research may provide strategies for the site’s selective amino acid modification of some natural peptides to achieve the desired enhancement of activity.Due to the abuse of traditional antibiotics and the continuous mutation of microbial resistance genes, microbial infections have become serious problems for human health. Therefore, novel antibacterial agents are urgently required, and amphibian antimicrobial peptides (AMP) are among the most interesting potential antibacterial leads. In this research, a novel peptide, named kassporin-KS1 , with moderate antibacterial activity against Gram-positive bacteria, was discovered in the skin secretion of the Senegal running frog,  Since the widespread availability of antibiotics beginning in the 1950s, antibiotics have been widely used clinically. Antibiotics can help to cure acute and refractory infectious diseases and promote the implementation of clinical treatments, such as chemotherapy and organ transplantation ,3. Thus,Antimicrobial peptides (AMPs) are derived from immune defense substances in most vertebrates . Most poKassina senegalensis, a kind of terrestrial frog, is widely distributed in sub-Saharan Africa [Kassina senegalensis contains potential biologically active peptides worth exploring through in-depth study.Amphibians are the main source of natural AMPs . Kassinan Africa . Many bin Africa ,13,14,15Kassina senegalensis are different. When these peptides were searched in peptide databases such as BLAST , there were no peptides that shared highly conserved sequences with them. Hence, the peptides from Kassina senegalensis possess unique sequences, and have not been classified into certain families. From the analysis of sequences and antibacterial results, some peptides in Kassina senegalensis show similarities to some temporin family peptides.Novel amphibian AMPs have been continually discovered over several decades . Most AMRana temporaria frog’s skin in 1996 [Temporins constitute one of the largest AMP families, and nearly 150 members have been described as of the end of 2020. Among the smallest AMPs in Nature, temporin was first obtained from the  in 1996 . They ha in 1996 .Through bioinformatics analyses of the members of the temporin peptide family, it was discovered that they have a high proportion of hydrophobic amino acids . From anMost temporins specifically inhibit Gram-positive bacteria by destroying their membranes at low concentrations (less than 20 µM) . This isKassina senegalensis. This temporin-like peptide has exhibited moderately potent inhibition of Gram-positive bacteria, and has the potential to be optimized by structural engineering into a highly effective analogue. The “glycine-lysine” motif from temporin peptides was incorporated at different sites into QUB-1641 to synthesize a group of such structurally modified analogues. The peptides were tested for their antibacterial efficacy, anti-biofilm effects, hemolysis effects, permeabilization of microbial membranes, and the systemic treatment of bacterially infected larvae compared to the synthetic replicate of the natural peptide.This study reports the isolation, structural and functional characterization of a novel AMP, named kassporin-KS1 (QUB-1641), from the skin secretion of the African hyperoliid frog, Kassina senegalensis skin secretion cDNA library by molecular cloning. The novel peptide-encoding precursor nucleotide sequence and the corresponding translated amino acid sequence are presented in The full-length cDNA encoding a novel peptide kassporin-KS1 (QUB-1641) was obtained from the Kassina senegalensis skin secretion was fractionated by RP-HPLC (The  RP-HPLC . As indi RP-HPLC a. Kasspo RP-HPLC b. The amThe sequence of kassporin-KS1 (QUB-1641) was searched using the Basic Local Alignment Search Tool (BLAST), and the results indicated that the novel sequence was not identical to any archived entry. However, through the use of the multiple sequence alignment program Clustal Omega, kassporin-KS1 (QUB-1641) was found to have some similarities to a few temporin peptides . They weAccording to the findings above, the structure of kassporin-KS1 (QUB-1641) was modified at different positions by inserting the “glycine-lysine” motif of the temporins. The effect of the modification on a range of bioactivities was studied using designed analogues, QUB-1599, QUB-1585, QUB-1570, and QUB-1643 . QUB-157The chromatograms of the synthesized peptides purified by RP-HPLC are shown in 4Ac solution presented a broad negative peak at 207 nm, indicating that the peptides all exhibited random coil structures in the aqueous environment. In 50% TFE/10 mM NH4Ac solution, all peptides showed a positive peak at 192 nm and negative peaks at 208 and 222 nm, indicating that the peptides exhibited α-helical structures in membrane-mimicking environments.The amino acid distributions and hydrophobic moments of QUB-1641 and its analogues are shown in S. aureus were eightfold more potent than QUB-1641. The modification of QUB-1570 was the most successful, and this peptide specifically eliminated Gram-positive bacteria at 8 µM.The antibacterial activities of kassporin-KS1 (QUB-1641) and its analogues against representative microbes are shown in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are shown in S. aureus biofilm growth. The MBICs of the analogues against S. aureus and E. coli were significantly higher than their MICs. QUB-1498 and QUB-1609 inhibited the S. aureus biofilm at 32 μM, but the mature S. aureus biofilm was only eliminated when the concentration reached 128 μM. Among all the peptides, QUB-1570 was the most effective in eliminating stubborn biofilms. QUB-1570 destroyed the S. aureus biofilm at 16 µM. Moreover, almost all peptides lost their activities on the biofilms of E. coli, but QUB-1570 was able to clear E. coli biofilm at 64 μM.The effects of kassporin-KS1 (QUB-1641) and its analogues on the biofilms of S. aureus. Thus, their bactericidal efficiency against S. aureus was tested  inhibited the growth of s tested . BesidesS. aureus membrane was investigated  were dosed after the larvae were infected with valuated . The pepAmphibian antimicrobial peptides (AMPs) have become the lead compounds expected to replace conventional antibiotics due to their low resistance generation and abundant sources . HoweverKassina senegalensis. Although the peptide was unique in sequence, it shared similarities with some temporins, such as displaying the “glycine-lysine” motif commonly found in temporins. Therefore, the core content of our research was to use this motif to optimize QUB-1641 and study structure-activity relationships. The motif “glycine-lysine” has been found to have positive effects on the antibacterial activity of AMPs.In this study, a novel biologically active peptide, kassporin-KS1 (QUB-1641), was identified in the skin secretion of From the composition of amino acids in the motif, the long side-chain of lysine helps improve the permeability of peptides in microbial membranes . In addiFor the glycine contained in the motif, due to its simple structure, its conformation has excellent stability. The presence of a single glycine could reduce the accumulation of conventional helical structures, but it can also interfere with a helical structure’s stability ,30. TherAfter that, the analogues QUB-1498 and QUB-1609 were synthesized based on the sequence of QUB-1570. QUB-1498 had replaced the lysine at position 9 with glycine. Therefore, QUB-1498 had one less positive charge, and its helical content was also significantly reduced. For QUB-1609, the cationic was retained, but the glycine at position 8 was replaced with proline. The helical structure of QUB-1609 was disrupted due to the presence of proline . In the Amino acid compositions, number of positive charges, and hydrophobicity affect the activity of AMPs ,34. In tStaphylococcus aureus and Escherichia coli are common ESKAPE pathogens that constitute biofilms [It is not appropriate to judge these peptides’ antibacterial effect as drugs based on the MIC and MBC values of the peptides in the planktonic state. The effect of antibacterial agents is usually reduced after microorganisms form biofilms . The biobiofilms . Once thbiofilms . For QUBbiofilms . QUB-157S. aureus at their MICs, indicating that the antibacterial mechanisms of QUB-1641 and modified peptides are related to the destruction of microbial membrane structure. The penetration ratio of QUB-1570 to S. aureus was higher than other analogues. Thus, QUB-1570 was speculated to cause microbial cell death by affecting microbial membranes, similar to most temporins [In the membrane permeability assay, all peptides permeabilized emporins .S. aureus, the time-killing kinetics of the peptides on S. aureus were also tested. Most peptides at the MICs did not completely kill S. aureus since the bacteria’s continuous growth rate exceeded the inhibitory efficiency of peptides on the microorganisms during the assay. All peptides were able to eliminate S. aureus at 2 × MICs. This was probably since the 2 × MICs of the peptides against S. aureus were close to their MBCs, achieving more effective killing. Among these, 2 × MIC of QUB-1570 eliminated S. aureus within 10 min. Combined with the membrane permeability assay results, it could be concluded that QUB-1570 achieves antibacterial effects by rapidly destroying microbial membranes.After exploring the peptides’ antibacterial mechanisms on S. aureus infection.Based on the discoveries above, the anti-biofilm activities of QUB-1570 might be attributed to its rapid elimination of bacteria, which could destroy the initially formed biofilm, prevent further accumulation of colonies, and accelerate the lysis of mature biofilms. QUB-1570 could be applied in targeted therapy of diseases related to However, the antimicrobial activity sensitivity of peptides at different physiological concentrations of ions restricted their development as potential clinical drugs. With the exceptions of QUB-1498 and QUB-1609, the peptides synthesized with the “glycine-lysine” motif showed poor antimicrobial activity in acidic, alkaline, or ionic environments. Their antibacterial activity was reduced by at least fourfold. This may be caused by the high sensitivity of lysine to pH changes .S. aureus by QUB-1498 and QUB-1609, which did not have the “glycine-lysine” motif. Magnesium ions have been reported to reduce the formation of biofilms and act synergistically with AMPs to promote greater curvature on their membranes. It affects the membrane structures of microorganisms, and ultimately leads to membrane leakage [Interestingly, the physiological concentration of magnesium ions weakly increased the inhibition of  leakage . Nonethe leakage . This imIn addition to assessing peptide antibacterial activities, peptide effects on erythrocytes as surrogate body cells should also be considered to measure possible cytolytic effects. The analogues’ antibacterial activity had improved, and most of them did not cause significant hemolysis within the MBC range for Gram-positive bacteria. QUB-1570 and QUB-1498 caused a higher proportion of hemolysis above 64 μM. This may be due to their helical content and hydrophobicity interfering with normal red blood cell membranes at high concentrations .S. aureus infected larvae to more than 50% at 24 mg/kg, close to the survival rate induced by the positive control. This showed that the most successfully modified peptide, QUB-1570, achieved decent efficacy in the initial treatment of animal models infected with S. aureus.In the in vivo assay, the larvae possessed innate immune systems similar to mammals, therefore they were the preferred infection model . QUB-157S. aureus-related infections. This peptide will be further modified to be closer to the standard of clinical application. On the other hand, it is necessary to further prove the effectiveness of the “glycine-lysine” motif by expanding its application scope, such as optimizing other short peptides.Overall, through the modification of the novel AMP, kassporin-KS1, by the “glycine-lysine” motif, the motif’s influence on structure-activity relationships was clarified, and opened up new ideas for the optimal design of short active peptides. On the one hand, the analogue QUB-1570, synthesized by replacing the eighth and ninth amino acid of kassporin-KS1 with “glycine-lysine”, had the best potential to treat Kassina senegalensis frogs were kept for four months before harvesting their skin secretions . The back of the frogs was gently massaged and transdermal electrically stimulated. Then, the skin secretions were flushed with deionized water into the beaker. After that, skin secretions were quickly lyophilized into powder by liquid nitrogen and stored in a −20 °C freezer. This experiment complied with the UK Animal (Scientific Procedures) Act 1986’s relevant regulations, and was reviewed by the Animal Care and Use Institutional Committee of Queen’s University Belfast on 1 March 2011.Kassina senegalensis was isolated. The isolated mRNA was reverse transcribed into cDNA using Dynabeads® mRNA DirectTM kit , and the corresponding cDNA library was set up by rapid amplification PCR (RACE-PCR). The complete peptide nucleotide sequence was achieved through the SMART-RACE cDNA amplification kit . Nested universal primers (NUP) and sense primers (AC-S AGC AGC AAA AGA AGA AGA AGC CAT G) were used in 3′-RACE reactions to obtain the target sequence. The purification and cloning of RACE products were then provided by the application of the pGEM®-T Easy Vector System . The nucleotide sequence of the cloned product was analyzed by the ABI PRISM® 3100 Genetic Analyzer .The polyadenylated mRNA of the lyophilized skin secretion of Kassina Senegalensis skin secretion was dissolved in 0.5 mL buffer (water/ trifluoroacetic acid (TFA), 99.95/0.5, v/v) and centrifuged. The supernatant was then pumped into the Reversed Phase High Performance Liquid Chromatography (RP-HPLC) with Jupiter® C18 Column . Skin secretions were eluted by a linear gradient program from water/TFA  to water/TFA/acetonitrile . The RP-HPLC ran at 1 mL/min for 240 min and automatically collected fractions every minute. The supernatant of the centrifuged skin secretions was also pumped into the LCQ Fleet Ion Trap Mass Spectrometer. Peptide masses and sequences were obtained through their mass-to-charge ratio and the dissociation fragments. Proteome Discoverer 1.0 software was applied to identify the mass and amino acid sequence of QUB-1641.Approximately 5 mg 2O, 2% 1,2-ethanedithiol (EDT), and 94% TFA. After the impurities were filtered, the peptide was precipitated, washed, and lyophilized. Then, the purified peptide and its analogues were collected by RP-HPLC, and the purity of the peptides was determined by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).Sufficient peptides involved in the research were synthesized by Fmoc Solid-phase peptide synthesis (SPPS) . Each amhttp://heliquest.ipmc.cnrs.fr, accessed on 25 October 2021). The peptides’ secondary structures were predicted by circular dichroism (CD) spectroscopy. The peptide samples were dissolved in 10 mM ammonium acetate (NH4Ac) and trifluoroethanol (TFE)/10 mM NH4Ac  to form 50 μM solutions. Then, the quartz cuvette containing the peptide solution was placed in the JASCO J-815 spectropolarimeter  at 20 °C for structural analysis at 190–250 nm . Finally, the sample value was obtained through three-pass scanning accumulation. Then, the helix ratio of these peptides was tested on BestSel .The amino acids and sequences of kassporin-KS1 (QUB-1641) and its analogues were analyzed by the online tool Heliquest , Enterococcus faecalis , Staphylococcus aureus , Klebsiella pneumoniae , Methicillin-resistant Staphylococcus aureus , Pseudomonas aeruginosa , and Escherichia coli .The inhibition of representative microorganisms by peptides was evaluated by the modified broth dilution technique. The microorganisms included 2 μM solution with phosphate-buffered saline (PBS) and then double-diluted into 64, 32, 16, 8, 4, 2, and 1 × 102 μM. Each sample solution and positive control (2 g/mL Norfloxacin) was added to 96-well plates and divided into five replicates, 1 μL per well. The blank control was 100 µL MHB, and the growth control was 100 µL bacterial solutions. When the optical density (OD) value corresponding to the microbial concentration reached the value  in their logarithmic growth phase, the microorganisms were diluted to the required concentration with MHB .Firstly, the selected microorganism was incubated in conical flasks with 100 mL Mueller Hinton broth (MHB) overnight. Then, the bacterial solution was sub-cultured in 20 mL MHB until it reached the logarithmic growth phase. Meanwhile, the peptide solution was prepared. The peptide was dissolved into 128 × 10The diluted bacterial solution was then added to the 96-well plate at 99 µL per well. The plate was incubated at 37 °C and observed the next day. The MIC was the corresponding lowest peptide concentration without microbial growth. The absorbance of the 96-well plate at 550 nm was measured by Synergy HT Microplate Spectrophotometer , and the obtained values were quantitatively analyzed by Prism 7 software. Subsequently, the concentrations corresponding to the clear solution were removed from the 96-well plate in 10 µL and dropped onto the solidified MHA. The Petri dishes with MHA were cultured overnight, observed and the minimum peptide concentration without colonies was MBC.Staphylococcus aureus  and Escherichia coli  was selected as representative strains. For the MBIC assay, the cultures were sub-cultured to their logarithmic growth phases. The diluted bacterial suspension was incubated with the peptide solution overnight in a 96-well plate. The resulting biofilm was washed with PBS solution, fixed with methanol, and dried. The biofilm was then stained with 0.1% (w/v) crystal violet, washed again with PBS, and dissolved in 30% acetic acid. The absorbance of the solution in each well at 595 nm was counted by the Synergy HT Microplate Spectrophotometer . Compared with the negative control, the lowest concentration that inhibits biofilm formation was MBIC. For the determination of MBEC, the diluted bacterial solution was incubated in the 96-well plate for 24 h, with 100 µL per well. Then, the biofilm was washed and re-incubated with 100 µL peptide-medium solution. After 24 h, the biofilm was again washed, stained, and dissolved as in the MBIC assay. Each well was detected at 595 nm by the same plate reader in MBIC. Compared with the negative control, the lowest concentration to eliminate biofilm was MBEC.As previously described, the MBIC and MBEC of the peptides against bacteria were determined . StaphylStaphylococcus aureus  was transferred from MHB to TSB for sub-culture until it reached its logarithmic growth phase. After centrifuging the bacterial suspension, the bacterial cells were collected. The bacteria were washed twice according to the steps of adding 5% TSB, centrifuging, and discarding the cell supernatant. Then, the washed bacteria were mixed with 5% TSB until the absorbance of the bacterial suspension at 590 nm reached the OD value of 1 × 108 CFU/mL. In total, 50 µL of bacterial suspension and 40 µL peptide solution were blended in each well of a light-shielded 96-well plate for 2 h at 37 °C. The positive control was the suspension of bacteria completely lysed with 70% (v/v) isopropanol. Each well was then filled with 10 µL SYTOX green. The fluorescence intensity of the wells was tested using a microplate spectrophotometer  at 485 nm excitation and 528 nm emission wavelengths.Staphylococcus aureus  was sub-cultured to the logarithmic growth phase and diluted to 5 × 105 CFU/mL. The peptide solution and the bacterial suspension were mixed into MIC, 2 × MIC, and 4 × MIC solutions. Then, the solution was aspirated at 10 µL from the tube at 0, 5, 10, 15, 30, 60, 90, 120, and 180 min, diluted with PBS, and dropped onto Petri dishes containing MHA. After incubating overnight, viable counts were performed on the cultured colonies.The cultured 2, and 4 μM FeCl3) and MHB with pH 6.0 and pH 8.0 were prepared. The selected microorganism Staphylococcus aureus  was incubated overnight in the prepared medium, sub-cultured to the logarithmic phase, and then diluted to 5 × 105 CFU/mL. The subsequent steps were carried out as the MIC/MBC assay.As mentioned in the previous study, the salt and pH sensitivities of AMPs exposed to physiological concentrations of salts or non-neutral environments were tested . MHB cong repeatedly until the supernatant turned clear. The PBS solution in the tube was finally replenished and mixed to form 4% (v/v) erythrocyte suspension. Meanwhile, peptides were diluted with PBS to form peptide solutions and then serially diluted to the lowest concentration. The negative control was PBS solution, and the positive control was 1% Triton X-100. Then, 200 µL of 4% erythrocyte suspension was added to the 1.5 mL tubes containing 200 µL peptide solution or the control and incubated at 37 °C. After two hours, the tubes were taken out and centrifuged at 900× g. Then, 100 µL of supernatant were removed to a new 96-well plate. Their absorbance at 550 nm was detected by the microplate spectrophotometer .As described previously , two mL Galleria mellonella larvae of equal weight were injected with bacterial suspensions to observe the peptides’ efficacy [S. aureus NCTC 10788 was cultured in sterilized saline until the bacterial suspension density reached 108 CFU/mL. The larvae injected with the bacterial suspension were observed for viability within 2 h. Then, 10 µL peptide solutions were injected into the larvae at 6, 12, and 24 mg/kg. The positive control was the dosage of 20 mg/kg Vancomycin. The negative control was the administration of saline. In total, nine wax worms were included in each group. After being injected, the number of larvae deaths was recorded every 12 h until it reached 120 h.efficacy . First, p < 0.05), ** (p < 0.01), *** (p < 0.001), **** (p < 0.0001) and ns (no significant difference).Graph Pad Prism 7.00 was applied to analyze all of the data . Error bars reflected the standard error (SEM) of five iterations. The larvae models were evaluated using the log-rank test. Significant differences were indicated by * (


Akkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The response of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs was examined in quiescent, lipopolysaccharide (LPS)-activated LX-2 cells. Liver fibrosis was induced in 8-week-old C57BL/6 mice, using a high-fat diet (HFD) and carbon tetrachloride (CCl4) administration for 4 weeks. The mice were concomitantly treated via oral gavage with three forms of bacteria. The relative expression of different fibrosis and inflammatory markers was assessed in the tissues. Histological markers, serum biochemical parameters, and cytokine production were also analyzed, and their correlations with the relative abundance of targeted fecal bacteria were examined. All A. muciniphila preparations exhibited protective effects against HSC activation; however, EVs showed the greatest activity in HSC regression. Oral gavage with A. muciniphila ameliorated the serum biochemical and inflammatory cytokines and improved liver and colon histopathological damages. The relative expression of fibrosis and inflammatory biomarkers was substantially attenuated in the tissues of all treated mice. The composition of targeted stool bacteria in the live A. muciniphila group was clearly different from that in the fibrosis group. This study indicated that A. muciniphila and its derivatives could successfully protect against HFD/CCl4-induced liver injury. However, further studies are needed to prove the beneficial effects of A. muciniphila on the liver.IMPORTANCEAkkermansia muciniphila, as a member of the gut microbiota, has been proposed as a next-generation probiotic. Liver fibrosis is the main determinant of liver dysfunction and mortality in patients with chronic liver disease. In this study, we aimed to determine the beneficial effects of live and pasteurized A. muciniphila and its extracellular vesicles (EVs) on the prevention of liver fibrosis. The results of the present study indicated that oral administration of live and pasteurized A. muciniphila and its EVs could normalize the fecal targeted bacteria composition, improve the intestinal permeability, modulate inflammatory responses, and subsequently prevent liver injury in HFD/CCl4-administered mice. Following the improvement of intestinal and liver histopathology, HFD/CCl4-induced kidney damage and adipose tissue inflammation were also ameliorated by different A. muciniphila treatments. Chronic liver disease (CLD) refers to liver dysfunction as a consequence of progressive destruction and regeneration of liver tissue, which can lead to fibrosis and hepatic carcinoma with high mortality rates . A groupDue to the increased development of new antiviral medications, as well as the increased prevalence of obesity and its related complications in recent decades, CLD is potentially becoming a nonviral disease worldwide . NAFLD, Akkermansia muciniphila, a bacterium that inhabits the gastrointestinal mucus layer, has been recently considered a next-generation probiotic strain in the treatment of obesity-related disorders (A. muciniphila-derived outer membrane proteins (OMPs), metabolites, and ligands with other members of the intestinal microbial community  are submicron-sized bilayer lipid structures that are derived from the cell membrane of both Gram-negative and Gram-positive bacteria and can interact not only with the host cells but also with other microbiota , 17. TheA. muciniphila-derived EVs seem to improve the gut barrier integrity in high-fat-diet (HFD)-induced diabetes by penetrating the large intestine and spreading to peripheral tissues, such as muscle, adipose, and liver tissues , colon tissue, and kidney tissue. The fecal bacterial quantification was also performed in stool samples obtained at the end of study at different taxonomic levels, using real-time PCR method in all groups. Finally, the effects of different forms of A. muciniphila on the HSC activation, fibrotic and inflammatory markers, and fecal targeted bacteria alterations were compared in vitro and in vivo.In the present study, we used a meta-analysis of seven previously collected carbon tetrachloride (CCl4)-induced liver injury microarray data sets –30 to id–iniphila . Next, tP value of <0.05 by the Mann-Whitney method. As shown in the heatmap . Moreover, ECM regulator genes involved in tissue fibrosis and inflammation  were significantly overexpressed in the fibrosis group compared to those in healthy control (HC) mice (P < 0.0001). Principal-component analysis (PCA) was applied for the selected genes between all 25 fibrosis and 25 HC samples from seven data sets. As shown in A. muciniphila on fibrosis-related markers. The results indicate that all eight genes were significantly differentially expressed between fibrosis groups in comparison with healthy control groups.The meta-analysis performed on data sets comparing mRNA levels in mice with CCL4-induced liver injury to those in controls shows that the expression of eight genes, including tumor necrosis factor alpha (TNF-α), TLR-2, TLR-4, transforming growth factor beta (TGF-β), alpha smooth muscle actin (α-SMA), platelet-derived growth factor (PDGF), tissue inhibitor of metalloproteinases (TIMP1), and collagen type 1 alpha 1 (Col1a1) in liver, are closely related to fibrosis and were differentially expressed, with a  heatmap , inflammA. muciniphila were evaluated by scanning electron microscopy (SEM); the results showed a spherical shape and a range in size of 40 to 150 nm  and EVs in various concentrations  for 24 h. As shown in Figure S2, in LPS-stimulated LX-2 cells, the highest inhibitory effects of live and pasteurized A. muciniphila on the gene expression of TLR-2  and TLR-4  were observed at MOI 10. In comparison with heat-killed A. muciniphila, which was examined in our previous study, pasteurized A. muciniphila showed lower mRNA level of TLR-4 at MOI 1 and 100 , but the effect of heat-killed and pasteurized A. muciniphila was almost the same at MOI 10. EVs at a concentration of 50 μg/ml significantly (P < 0.0001) downregulated TLR-2 and TLR-4 expression. In the quiescent LX-2 cells, although all A. muciniphila treatments increased the expression of TLR-2 and TLR-4, this increase was not statistically significant . In quiescent cells, only EVs at a concentration of 50 μg/ml could downregulate the expression of TLR-2 gene . Overall, consistent with data obtained from het-killed A. muciniphila, these data also suggest that the effects of live and pasteurized A. muciniphila on quiescent and LPS-activated LX-2 cells were not dose dependent, as these two forms showed their best effect at MOI 10, while in EVs, we observed an improvement in its effect by increasing the concentration. The most effective bacterial MOIs and EVs concentration are shown in To investigate whether A. muciniphila and its EVs on gene expression of TGF-β, α-SMA, TIMP1, and Col1a1 in LX-2 cells is shown in Figure S2. In the quiescent LX-2 cells, there was no significant difference between expression of fibrosis markers in treated mice and untreated control. In LPS-stimulated LX-2 cells, the expression of fibrosis markers was dramatically increased (P < 0.0001). Pasteurized A. muciniphila could downregulate the expression of fibrosis markers at MOI 10 (P < 0.05), and in the case of TGF-β and α-SMA, this inhibitory effect was also seen at MOI 100 (P < 0.05). Almost all MOIs of live A. muciniphila significantly reduced the gene expression of fibrosis markers (P < 0.001); in the case of collagen, this reduction was observed only at MOI 10. Compared to our previous study, pasteurized A. muciniphila showed an mRNA level of fibrosis markers lower than that of heat-killed A. muciniphila , but the optimum dose of heat-killed and pasteurized A. muciniphila was the same (MOI 10). The inhibitory effects of EVs on expression of fibrosis markers were dose dependent, and the highest antifibrosis property was observed at a concentration of 50 μg/ml (P < 0.0001). These observations suggest that EVs could more efficiently reverse the activation of HSCs. The most effective bacterial MOIs and EVs concentrations are shown in The effect of live and pasteurized A. muciniphila (Am), pasteurized A. muciniphila (Pam), and A. muciniphila’s extracellular vesicles (EVs) , as shown in A. muciniphila, no significant difference was observed between the effects of these two groups on body weight , liver weight (P > 0.99), and liver/body weight ratio (P > 0.99). Histopathological analysis confirmed that acute liver injury was established in the PBS group compared to that in the normal diet (ND) group (A. muciniphila (Pam) groups, while almost no effect of fibrosis induction was observed in live A. muciniphila (Am) and extracellular vesicles (EV) groups. Pericellular fibrosis occurred around damaged liver hepatocytes in the liver parenchyma, as well as around the portal portion of the liver in the PBS and pasteurized A. muciniphila (Pam) groups. Also, the presence of small and large droplets inside hepatocytes was observed in the PBS group compared to other groups. Moreover, we evaluated the effects of all bacterial treatments on pathology of colonic tissue. The PBS group showed the focal infiltration of inflammatory cells  in the lamina propria and epithelium in mice colonic tissue. No inflammatory reaction was present in live A. muciniphila (Am) and extracellular vesicles (EV) groups. The crypt depth and thickness of the mucous layer of the colon showed a considerable decrease in the PBS group compared to that in the normal diet (ND) group. The crypt depth and thickness of the mucous layer showed an increase in pasteurized A. muciniphila (Pam), live A. muciniphila (Am), and extracellular vesicles (EV) groups compared to those in the PBS group  . These mD) group . HematoxBS group .A. muciniphila, pasteurized A. muciniphila, and A. muciniphila’s EVs, the serum level of TNF-α, interleukin 6 (IL-6), and Il-10 was assessed. As shown in P < 0.0001) and IL-10 significantly decreased  in PBS compared to those in the ND group. Interestingly, all treatments could significantly decrease serum level of TNF-α  and IL-6 (P < 0.0001) while increasing IL-10 . Interestingly, these anti-inflammatory effects were more pronounced in EV than in other groups, suggesting that A. muciniphila may inhibit liver fibrosis through modulating of the serum cytokines, which is more evident in EVs.To investigate anti-inflammatory effects of live P < 0.0001). Daily oral gavage with all bacterial treatment was able to decrease serum level of ALT and AST, while in Am and EV groups, this improvement was much more pronounced (P < 0.0001) than that in the Pam group. As shown in P value of 0.01), total cholesterol , total triglyceride , low-density lipoprotein , and very-low-density lipoprotein , while in the serum level of high-density lipoprotein , it had the opposite effect in the PBS group compared to that in the ND group. Administration of two forms of whole cell bacteria and EVs reduced blood glucose, especially in the Am group (live bacteria) compared to that in the PBS group . Also, a significant decrease in the serum level of TG and TC was observed in Am and EV in comparison with that in the PBS groups , whereas TC level did not show a significant difference in the Pam group compared to that in the PBS group , and the effect of A. muciniphila on TG was also less significant than that of the other forms used in this study . In the case of LDL, all bacterial forms and EVs significantly (P < 0.0001) decreased the LDL compared to that of the PBS group. Although VLDL was significantly reduced in all groups, Am and EV showed the highest decrease compared to PBS . Surprisingly, the highest incremental effect on HDL was related to EV between the groups (P < 0.0001). Overall, these results suggest that A. muciniphila and its EVs could prevent liver fibrosis by normalizing serum glucose, lipid profiles, and liver enzymes.Evaluation of serum levels of liver enzymes showed a significant increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of the PBS group compared to those of the ND group . The gene expression analysis showed that live A. muciniphila and its EVs modulated the expression of α-SMA, PDGF, TIMP1, TGF-β, and Col1a1 genes (P < 0.0001). As demonstrated in P value of 0.001). These results suggest that administration of live and pasteurized A. muciniphila and its EVs could reduce hepatic damage induced by HFD/CCl4 through inhibiting the expression of α-SMA, PDGF, TIMP, and Col1a1 genes.To explore the protective effects of A. muciniphila on the inflammatory status of liver tissue, we assessed the expression of TLR-2, TLR-4, and TNF-α genes in HFD/CCl4 mice. A significant increase was observed in gene expression of these inflammatory markers in the PBS group compared to that in the ND group (P < 0.0001). As shown in P < 0.0001). This inhibitory effect was similar in all study groups, indicating that various forms of A. muciniphila could downregulate the expression of inflammatory genes in liver tissue and consequently inhibit hepatic injury induced by HFD/CCl4.To investigate the influence of A. muciniphila and its EVs could enhance the expression of ZO-1 mRNA in the colon tissue , while in the PBS group it was significantly downregulated . The TLR-2 was another factor that was upregulated in all treatment groups compared to that in PBS; however, the highest increase was observed in the Am group  (P < 0.0001)  . The exp 0.0001) . It can P < 0.0001). Interestingly, we found that the all forms of A. muciniphila and its EVs had an inhibitory effect on these fibrosis gene markers in the colon. However, live A. muciniphila and its EVs downregulated the expression of α-SMA and TGF-β (P < 0.0001) more significantly than did the pasteurized form . In the case of PDGF, all forms of A. muciniphila and its EVs demonstrated significant inhibitory effects (P < 0.0001). Overall, the results suggested that A. muciniphila and EVs could downregulate the fibrosis markers not only in the liver but also in the colon tissue , while the anti-inflammatory IL-10 was decreased (P < 0.0001). Our results demonstrated that live A. muciniphila and its EVs could downregulate the gene expression of α-SMA and IL-6 and upregulate the IL-10 (P < 0.0001) more significantly than could pasteurized A. muciniphila  . In the PBS group, fibrosis genes, including α-SMA, PDGF, and TGF-β, and proinflammatory genes, such as TNF-α and IL-6, were overexpressed (ctively)  to M. ThA. muciniphila could have antifibrosis activities on the kidney tissue, we evaluated α-SMA, PDGF, Col1a1, and TGF-β mRNA expression levels between study groups. As demonstrated in P < 0.0001). However, although all bacterial treatments significantly reduced the gene expression of these markers (P < 0.0001), the impact of pasteurized A. muciniphila on TGF-β downregulation was less significant than that of other treatment groups . Overall, the results showed that all three bacterial forms used had a significant preventive effect on the HFD/CCL4-induced injury in kidney tissue.In order to investigate whether the EVs or various forms of A. muciniphila and its EVs on the intestinal microbial community was investigated  and increased Bacteroidetes  relative abundance in the PBS group compared to that in the ND group , while administration of live A. muciniphila could considerably restore this imbalance (61.67% for Firmicutes versus 31% for Bacteroidetes). The effect of pasteurized A. muciniphila and its EVs on Firmicutes and Bacteroidetes abundance was not statistically significant . The Firmicutes to Bacteroidetes (F/B) ratio was significantly  lower in the PBS group than in the ND group. In addition, the F/B ratio in the Am group was significantly  higher than that in the PBS group. As shown in A. muciniphila and EV groups was higher than that in the PBS group, but this difference was not statically significant. Moreover, HFD/CCl4 significantly increased the relative abundance of Fusobacteria and decreased Actinobacteria in the PBS group compared to those in the ND group , while only live A. muciniphila could significantly balance these two phyla . As shown in Gammaproteobacteria , Epsilonproteobacteria , and Enterobacteriaceae  at the class/family level in the PBS group, while live A. muciniphila was able to significantly increase Clostridia , Ruminococcaceae , and Prevotellaceae . At the genus/species level, Lactobacillus spp. and Escherichia coli were enriched in PBS compared to those in the ND , while Bifidobacterium spp., Roseburia, Methanobrevibacter, Alistipes, Veillonella, and Faecalibacterium prausnitzii were more abundant in the ND group  and the Am group  than in the PBS group , which indicated that live bacteria colonization occurred successfully after 4 weeks of oral gavage. The pasteurized A. muciniphila and its EVs also possibly induced the A. muciniphila growth after oral gavage. The pasteurized A. muciniphila could also modify the intestinal targets microbiota of mice in the case of Gammaproteobacteria, Veillonella, and Enterococcus . Moreover, the EVs positively altered the mice intestinal targets’ microbiota compared to that of the PBS group in the case of Gammaproteobacteria and Enterococcus ; however, other targets’ microbiota changes were not statistically significant. These data demonstrate the ability of live A. muciniphila to reduce the relative abundance of pathobionts and increase symbionts, therefore shifting the targets’ microbiome structures to be similar to those of the ND group.Since an HFD affects gut bacterial composition and induces intestinal dysbiosis, the effect of live, pasteurized stigated . HFD/CCLBS group . As expeEnterococcus spp., which were more abundant in the PBS group, positively correlated with serum level of TG , TC, LDL, and Glu . The serum level of TC and LDL was also positively correlated with Lactobacillus spp. . Moreover, Escherichia coli showed positive association with TC . Conversely, TC was negatively associated with beneficial bacteria such as A. muciniphila and Bifidobacterium spp. , which were more abundant in the ND and Am groups. Bifidobacterium spp. also showed negative association with LDL . Enterococcus spp. , Lactobacillus spp. , and E. coli  also demonstrated positive correlation with serum proinflammatory cytokines, while they inversely associated with anti-inflammatory cytokine IL-10 . ALT, as one of the most important blood biomarkers in liver injury, was negatively related to Methanobrevibacter and Bifidobacterium spp.  and was positively associated with E. coli and Lactobacillus spp. . Another liver enzyme, AST, was inversely related to Methanobrevibacter and Bifidobacterium spp. and A. muciniphila , while it was positively related to Enterococcus spp., Lactobacillus spp., and E. coli . Likewise, the body weight also demonstrated positive correlation with Enterococcus spp., Lactobacillus spp., and E. coli , while it demonstrated negative correlation with Bifidobacterium spp. . These data proposed the beneficial impact of gut microbiota on modulating the host response against the HFD/CCl4 challenge.To further explain the impact of gut microbiome structure, we adopted correlation analysis between the relative abundance of bacteria at the genus/species level assessed in this study and blood biomarkers. As shown in A. muciniphila, as a next-generation probiotic with beneficial effects on the host metabolic system and immune responses, has been introduced as a promising therapeutic option for microbiota-related diseases . This serum biochemical adjustment was also associated with the decreased level of proinflammatory cytokines (IL-6 and TNF-α) and increased production of anti-inflammatory cytokines (IL-10).Serum levels of transaminases (ALT and AST) are associated with the severity of liver histopathology. Evidence shows that an elevated ALT/AST level can result in a higher grade of liver injury . SystemiA. muciniphila for 10 weeks could reduce the serum TG and ALT levels in HFD-fed obese mice , and leptin and reduce the fat mass in HFD-fed mice  in the intestinal environment , the ATCC strain (similar to the strain of the present study) was associated with an increase in the production of SCFAs in a dextran sulfate sodium (DSS)-induced chronic murine colitis model, although both strains showed anti-inflammatory effects and normalized the gut microbiota  and downregulating TGF-β, while the effect of EVs was more noticeable  improved glucose tolerance, hyperlipidemia, and liver steatosis in HFD-fed mice  . DysbiosA. muciniphila reported in this study, further in-depth investigations are required to identify the mechanisms through which A. muciniphila may promote the kidney function and health. Consistent with our results, Yang et al.  was used to assess the expression profiling in case and control samples  database at the National Center for Biotechnology Information . Our inc samples , 84. TheAkkermansia muciniphila MucT (ATCC BAA-835) was obtained from the DSMZ institute (German collection of microorganisms and cell cultures). The bacterium was cultured anaerobically in a basal mucin-based medium at 37°C for 3 to 7 days (600) of 1 was reached. Bacterial pellets were harvested by centrifugation  and washed twice with sterile anaerobic PBS for cell culture treatment and oral administration in mice.o 7 days . After tA. muciniphila at an OD600 of 1. Then, the bacterial pellets were washed twice as mentioned above, and after that, the bacterial suspension was heated at 70°C for 30 min, as described previously  for EVs isolation. EVs were extracted with ultracentrifuge  as described previously . Cells were seeded in 24-well plates and were incubated in a 5% CO2 humidified atmosphere at 37°C.The LX-2 cell line, a spontaneously immortalized human HSC line, was kindly gifted from Scott L. Friedman . LX-2 cells were maintained in complete Dulbecco’s modified Eagle’s medium supplemented with 2 mM Escherichia coli 0111:B4 (Sigma-Aldrich) were used at a concentration of 0.01 μg/ml for 6 h  and with EVs of various concentrations  for 24 h in the CO2 incubator at 37°C. The LPS-activated LX-2 cells and untreated cells served as the control groups. Each experiment was performed in duplicate and repeated at least three times.The culture medium of the LX-2 cell monolayers of 80 to 90% confluence was replaced with fresh serum-free medium overnight. For LX-2 activation, the lipopolysaccharides (LPS) from  for 6 h . Afterwax × 100%)/y, where “x” is the absorbance of treated cells and “y” the absorbance of untreated cells.MTT  assay was carried out using the cell proliferation kit I (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, LX-2 cells were seeded in 96-well plates and treated separately with the aforementioned MOIs and concentrations of bacteria and EVs for 24 h. The percentage of cell viability was calculated using the following formula: cell viability (%) =  following the supplier’s protocol. The quantity and quality of RNA were verified via agarose gel electrophoresis and NanoDrop spectrophotometer . The purified RNA was reverse-transcribed to cDNA using the BioFACT RT-kit  according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) amplification was performed on a Rotor-Gene Q  real-time PCR system by using BioFACT 2× real-time PCR master mix , and GAPDH  served as the internal control. The primers used in this study are presented in Table S1.n = 25) and sterile hardwood chip bedding during the experiment. This study followed the institutional guidelines regarding the care and use of laboratory animals, and all animal experiments were designed in order to minimize mice suffering. The study protocol was approved by the Animal Experiment Committee Pasteur Institute of Iran (IR.PII.REC.1399.029).Male wild-type C57BL/6 mice were obtained at the age of 7 to 8 weeks from Pasteur Institute of Iran (Tehran Iran) and housed five mice per cage for the acclimatization period. Adaptation was performed in equal conditions  with free access to a standard normal diet (ND)  and autoclaved water during acclimation and throughout the study. All mice were individually housed in autoclaved cages  twice a week for 4 weeks (n = 5); healthy control animals received the ND only without any intervention, and Am (109 CFU/200 μl live A. muciniphila), Pam (109 CFU/200 μl pasteurized A. muciniphila), EVs (50 μg protein/200 μl), and vehicle (200 μl sterile PBS) were given by daily oral gavage for 4 weeks  was used for induction of liver injury in this study . The HFD 4 weeks . Mice weLiver and colon specimens were immediately fixed in 10% neutral buffered formalin after collection. Paraffin-embedded tissues were stained with hematoxylin and eosin (H&E) and Masson’s trichrome for liver and H&E in colon tissue. Microscopic observation of the histological slides was performed using a light microscope . StainedFasting blood glucose (Glu), total cholesterol, triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), very-low-density lipoprotein (VLDL), alanine transaminase (ALT), and aspartate transaminase levels were measured using a commercial kit  in the serum samples to evaluate the liver function.The level of serum IL-6, IL-10, and TNF-α was determined by using ZellBio GmbH enzyme-linked immunosorbent assay (ELISA) kit (Germany) according to the manufacturer’s instructions and analyzed with Bio-Plex Manager 6.1 software . The cytokine assays were performed in duplicate.Total protein was prepared from liver tissues using radioimmunoprecipitation assay (RIPA) lysis buffer. Cell lysates were centrifuged at 12,000 rpm for 20 min, and protein concentrations were measured using the bicinchoninic acid (BCA) protein assay . Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using 3% skim milk powder at 4°C overnight and then hybridized with the primary antibodies targeting the proteins, including GADPH and α-SMA . For the appearance of the protein bands, they were incubated with secondary antibodies horseradish peroxidase (HRP) conjugate goat anti-mouse IgG1  for 1 h at room temperature. The blotting bands were visualized using the ECL system .Frozen liver, colon, adipose, and kidney tissues were homogenized in 1 ml of TRIzol  using a Precellys 24 homogenizer, and the total RNA was extracted according to the manufacturer’s instructions. The genomic DNA was removed using DNase I (Qiagen), cDNA was synthesized, and qRT-PCR amplification was performed as mentioned earlier. The relative expression of target genes was assessed using the comparative cycle threshold (Ct) method. The RPL-19 and HPRT served as the normalizer genes for tissue samples.Fresh fecal samples were collected in sterile cups, immediately aliquoted in cryotubes, and stored at −80°C. Total bacterial DNA was extracted from 180 mg of each fecal sample using QIAamp DNA Stool minikit  according to the manufacturer’s protocol. The DNA purity and concentration were determined by Nanodrop spectrophotometer . The extracted DNAs were stored at −20°C until further microbiota analysis.Quantitative real-time PCR was performed in triplicate using Rotor-Gene Q  and SYBR green master mix  using specific primer pairs presented in Table S2. The amplification efficiency of each primer was determined by using the ΔCt method. The thermal cycling conditions were as follows: an initial DNA denaturation step at 95°C for 15 min, 40 cycles of denaturation at 95°C for 20 s, primer annealing at 56°C for 30 s, and extension at 72°C for 20 s. The average Ct value obtained from each reaction was transformed into percentage formula as described previously .post hoc test (for multiple comparisons between more than two groups) or two-way ANOVA where needed. Results are presented as the mean ± standard error of the mean of at least three experiments. The mean relative percentage of 16S rRNA genes was analyzed with the nonparametric Kruskal-Wallis test for comparison of the different groups. Spearman’s rho nonparametric correlation was performed to analyze relationships between relative abundance of microbiota, blood inflammatory and biochemical markers, and body/liver weight. These correlations were graphically visualized using Cytoscape v3.8.3  was used to calculate changes in gene expression and cytokine production. Differences between groups were calculated using one-way analysis of variance (ANOVA) followed by Tukey’s e v3.8.3 . A P val


Aims: Long-term salt diet induces the oxidative stress in the paraventricular nucleus (PVN) and increases the blood pressure. Extracellular superoxide dismutase (Ec-SOD) is a unique antioxidant enzyme that exists in extracellular space and plays an essential role in scavenging excessive reactive oxygen species (ROS). However, the underlying mechanism of Ec-SOD in the PVN remains unclear.Methods: Sprague–Dawley rats (150–200 g) were fed either a high salt diet  or normal salt diet  for 6 weeks. Each group of rats was administered with bilateral PVN microinjection of AAV-Ec-SOD (Ec-SOD overexpression) or AAV-Ctrl for the next 6 weeks.Results: High salt intake not only increased mean arterial blood pressure (MAP) and the plasma noradrenaline (NE) but also elevated the NAD(P)H oxidase activity, the NAD(P)H oxidase components (NOX2 and NOX4) expression, and ROS production in the PVN. Meanwhile, the NOD-like receptor protein 3 (NLRP3)–dependent inflammatory proteins  expression and the tyrosine hydroxylase (TH) expression in the PVN with high salt diet were higher, but the GSH level, Ec-SOD activity, GAD67 expression, and GABA level were lower than the NS group. Bilateral PVN microinjection of AAV-Ec-SOD decreased MAP and the plasma NE, reduced NAD(P)H oxidase activity, the NOX2 and NOX4 expression, and ROS production, attenuated NLRP3-dependent inflammatory expression and TH, but increased GSH level, Ec-SOD activity, GAD67 expression, and GABA level in the PVN compared with the high salt group.Conclusion: Excessive salt intake not only activates oxidative stress but also induces the NLRP3-depensent inflammation and breaks the balance between inhibitory and excitability neurotransmitters in the PVN. Ec-SOD, as an essential anti-oxidative enzyme, eliminates the ROS in the PVN and decreases the blood pressure, probably through inhibiting the NLRP3-dependent inflammation and improving the excitatory neurotransmitter release in the PVN in the salt-induced hypertension. In the brain, excess dietary salt increases the concentration of sodium in the brain that leads to endogenous ouabain (EO) release while the EO inhibits the Na+/K+-ATP pump and increases angiotensin II (ANG II) in the central system, which significantly elevates the sympathetic excitation and the blood pressure  intake elevated the reactive oxygen species (ROS) production and restrained the antioxidant capacity at once, which would cause the target organ damages such as vascular endothelial cell, renal tubules, myocardial performance, and central nervous system . As we kpressure . TherefoNumerous studies have reached an agreement that high salt diet leads to oxidative stress including the peripheral organs and central system ; 1999. IAs for the inflammation, there are numerous evidences showing that oxidative stress induced the inflammation in the PVN  and infll-tyrosine to l-3,4-dihydroxyphenylalanine (L-DOPA), is the initial and rate-limiting step in the biosynthetic pathway of catecholamines including dopamine, NE, and adrenaline. NE is released predominantly from the ends of sympathetic nerve fibers and acts to increase the force of skeletal muscle contraction and the rate and force of contraction of the heart so as to increase the blood pressure. Therefore, the high level of TH can increase catecholamine production, especially NE, which elevates the sympathetic activity and blood pressure. As we know, glutamic acid decarboxylase (GAD) is the crucial enzyme involved in the synthesis of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter of the central nervous system. Therefore, once the neurons were stimulated by acute or chronic factors like ROS, the resting potential of neurons were triggered to conduct the action potentials and transmits the neurotransmitters between prominences increasing the excitatory  release and suppressing the inhibitory neurotransmitter secretion such as GABA. Also, those excitatory neurotransmitters are released predominantly from the ends of sympathetic nerve fibers and then augmented the sympathetic nerve activity. All of those made us to explore whether the ROS in the PVN can regulate the neurotransmitters and finally decrease the sympathetic nerve activity and blood pressure in salt-induced hypertension.In addition, paraventricular nucleus of the hypothalamus exists in the presympathetic neurons and is a primitive action for modulating the downstream sympathetic nerve activity, which is tightly linked to blood pressure . Numerouvia regulation of the NLRP3 pathway and neurotransmitters in salt-induced hypertension.In summary, high salt diet induced the oxidative stress in the PVN, but how Ec-SOD regulates the sympathetic nerve activity and blood pressure remains unclear. Some researchers have indicated that ROS is closely related to inflammatory cytokines and neurotransmitters and probably takes part in the development of hypertension. Therefore, the aim of this study is to determine whether the overexpression of Ec-SOD in the PVN decreases the blood pressure ad libitum. The study was approved by the Animal Care and Use Committee of Xi’an Jiaotong University (No. 2018-404). The design conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health .Sprague–Dawley rats weighing 150–200 g were purchased from the experimental animal center of Xi’an Jiaotong University. All animals were housed in temperature-controlled (23 ± 2°C) and light-controlled (12 h light/dark cycle) animal quarters and were provided with rat chow Rattus norvegicus Ec-SOD targeting sequence  and green fluorescent protein was provided by Hanbio Biotechnology Co. Ltd . The related information is in 12 μg/ml and the serotype is AAV 9. After we obtained the adenovirus-associated virus, the AAV would be subpackaged (200 μl/tube) and store at −80°C. Before bilateral paraventricular nucleus injection of AAV-Ec-SOD or AAV-Ctrl, the vectors should be dissolved on ice.The Ec-SOD plasmid vector (HBAAV2/9-CMV-MCS-Zsgreen) containing the gene of For vector injection, rats were anesthetized with a ketamine (80 mg/kg) and xylazine (10 mg/kg) mixture , and the head was placed into a stereotaxic apparatus. The microinjector (5 μl) with vectors (either AAV-Ec-SOD or AAV-Ctrl) was installed in the microinjection pump. Vectors were bilaterally injected into the PVN  using 2 μl volume each side at the rate of 0.2 μl/min . After fSprague–Dawley rats (150–200 g) were fed with 8% high salt diet  and 0.9% normal salt diet  for 6 weeks. Then those two groups were respectively separated into AAV-Ec-SOD and AAV-Ctrl (empty vector) groups. Each group of rats was administered with bilateral PVN microinjection of the overexpression of AAV-Ec-SOD or AAV-Ctrl once at the beginning of the seventh week. High salt diet or normal salt diet was continued for a total of 12 weeks  and the via tail-cuff instrument, and their recording system has been described previously .For diaminobenzidine (DAB) staining, sections stained with primary antibody c-fos  were washed in PBS containing 0.1% Tween 20 for 1 h, then incubated with anti-rabbit IgG (HRP) secondary antibody  in blocking buffer for 1 h. The horseradish peroxidase reaction was detected using 3,3-diaminobenzidine (DAB) kit  according to the manufacturer’s instructions. Processing was stopped with HThe level of superoxide anion in the PVN was detected by dihydroethidium . Brain sections  were incubated for 10 min with dihydroethidium  at 37°C in the dark, as previously described. The oxidative fluorescence intensity was detected at 585 nm wavelength by fluorescence microscope imaging system (Nikon).As for the western blotting procedures, many references have been described . MicrodiAnalysis of relative change in gene transcripts was performed by qPCR assay. The hypothalamic tissues were dissected as described previously. Briefly, rat brains were isolated and cut into a coronal segment (−0.92 to −2.13 mm posterior to bregma). A block of the hypothalamus containing PVN was excised from the coronal section. Total RNA was extracted from microdissected PVN using Tri-Zol reagent  and reverse transcribed using oligo (dT) with conditions at 23°C for 10 min, 37°C for 60 min, and 95°C for 5 min. The cDNA was used for real-time PCR with specific primers for Ec-SOD  and GAPDH . The quantitative fold changes in mRNA expression were determined relative to glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA levels in each corresponding group.We used ELISA kits to measure plasma level of noradrenaline  . OxidatiWe used the HPLC-EC system to measure the level of GABA in the PVN, and the specific procedures had been described previously . Brieflypost hoc test was used to determine statistical differences in the number of positive neurons, fluorescent intensities, western blotting data, HPLC-EC, and ELISA tests.Data are presented as the mean ± SEM. Statistical analyses were performed using Prism version 8.0. MAP was analyzed by repeated measures ANOVA. One-way ANOVA followed by Tukey’s via tail-cuff instrument and plasma noradrenaline (NE) by ELISA. The results showed that excessive salt intake increased MAP from the second week. Until the sixth week, the MAP was higher in salt-induced group than in normal salt group . Overexpression of Ec-SOD in the PVN significantly decreased the MAP of salt diet group at the end of the 12th week  (p < 0.05). Overexpression of Ec-SOD in the PVN decreased the plasma NE level in hypertensive group (p < 0.05)  and sympathetic activity, we measured the MAP  < 0.05) . As for  < 0.05) .p < 0.05). After bilateral PVN microinjection of AAV-Ec-SOD, the PVN level of superoxide anion was lower than the high salt intake group (p < 0.05)  .p < 0.05). After bilateral PVN microinjection of AAV-Ec-SOD, overexpression of Ec-SOD significantly decreased the PVN expression of NOX2 and NOX4 in the salt-induced hypertensive group (p < 0.05). As for the Ec-SOD expression in the PVN, bilateral PVN microinjection of AAV-Ec-SOD is significantly higher than AAV-Ctrl (p < 0.05)  .p < 0.05). Overexpression of Ec-SOD significantly decreased the NAD(P)H oxidase activity and MDA in the salt-induced hypertensive group (p < 0.05). As for the GSH level in the PVN, bilateral PVN microinjection of AAV-Ec-SOD in the salt-induced hypertensive group increased the GSH level compared with AAV-Ctrl (p < 0.05) (p > 0.05) H oxidase activity, MDA level, and GSH level in the PVN by ELISA. Results indicated that NAD(P)H oxidase activity and MDA level in the PVN were higher in the excessive salt intake group than in the normal salt group ( < 0.05) . In addi > 0.05) .p < 0.05)  .p < 0.05)  . Meanwhip < 0.05). Overexpression of Ec-SOD in the PVN significantly decreased the expression of ASC, VCAM-1, pro-casp-1, CXCR3, IL-1β, and CCL2 in the salt-induced hypertensive group (p < 0.05)  .p < 0.05)  . It alsop < 0.05)  .p < 0.05). Overexpression of Ec-SOD in the PVN significantly decreased the TH expression in the PVN in the hypertensive group (p < 0.05). As for the GAD67 expression in the PVN, excessive salt intake decreased the PVN level of GAD67, but bilateral PVN injection of AAV-Ec-SOD significantly elevated the GAD67 expression in the PVN in salt-induced hypertensive group (p < 0.05)  .Long-term high salt augmented the NADP(H) oxidase-dependent ROS in the PVN and increased the blood pressure. Meanwhile, excessive sodium intake elevated the NLRP3-dependent inflammatory and broke the balance of neurotransmitters in the PVN. Ec-SOD, as an essential anti-oxidative enzyme, eliminated oxygen free radicals and decreased the blood pressure, probably through inhibition of the NLRP3-dependent inflammation and reduction of the excitatory neurotransmitter release in the PVN in salt-induced hypertension.+/K+ exchange mechanism on the cell membrane, and probably activates the downstream ROS signaling pathway –ANG II neuromodulatory pathway in the central system H-oxidase components.In this study, after injection of overexpression Ec-SOD, we also found that the expression of NLRP3 in the PVN is lower than in the hypertensive group. NLRP3 is one of (LRR)-containing (NLR) family members and is also the classic pathway that activates the inflammatory responses and inflammasome. There are many pathways or molecules such as TLR4/NF-κB pathway, ATP, poretoxin, and mitofusin2 (Mfn2), which can trigger the NLRP3-dependent inflammation . ActivatIn addition, the PVN of the hypothalamus is reciprocally connected to other areas of the central nervous system that are involved in cardiovascular function  and is a2·−) can combine with nitric oxide (NO) to form the peroxynitrite anion (ONOO−), which has an irreversible effect on their transportation and physiological functions H oxidase–dependent ROS in the PVN, activates the NLRP3-dependent inflammatory reaction, and increases excitatory but suppresses inhibitory neurotransmitters in the PVN. The overexpression of Ec-SOD in the PVN has a positive effect on the decrease of sympathetic activity and blood pressure probably through inhibition of the NLRP3-dependent inflammation and elevation of the inhibitory neurotransmitter release in the PVN in salt-induced hypertension .